Pseudotyped lentiviral vectors for gene therapy: impact of envelope glycoproteins

• Autor(a) principal: Nogueira, Rodrigo José Gameiro
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/89669
• topic: Gene therapy; Lentiviral vectors; Envelope glycoproteins; Stable expression; Constitutive producer cell lines; Terapia génica; Vetores lentivirais; Glicoproteínas do invólucro; Expressão estável; Linhas celulares produtoras constitutivas; Domínio/Área Científica::Ciências Médicas

The potential of gene therapy to treat a broad range of diseases, ranging from cancer to monogenic diseases, makes it a powerful approach to address medical needs that are currently unmet by conventional medicine. Lentiviral vectors have been the viral vectors experiencing the highest growth in gene therapy clinical trials due to their large genetic payload, the ability to transduce both dividing and non-dividing cells, permanently integrating into the target cell genome and increased safety when compared to Gammaretrovirus. However, the utilization of lentiviral vectors in gene therapy has been conditioned by the currently available production systems. The development of constitutive producer cell lines will provide a better alternative to cope with the increasing necessity of large-scale production of lentiviral vectors. This work focused on the evaluation of GaLV10A1ΔR envelope glycoprotein potential for stable expression, in order to enable its use in constitutive producer cells. This was based on a previously developed cell line, HEK 293T SLC20A1 knock-out, especially designed to cope with the cytotoxicity of GaLV10A1ΔR. To achieve stable expression of GaLV envelope glycoproteins new plasmids were constructed. An analytical method for syncytia quantification, based on cell size distribution, was also successfully developed. This work showed that SLC20A1 deletion enabled GaLV10A1ΔR expression, eliminating the cytotoxicity associated with its expression. More importantly, SLC20A1 knock-out enabled the establishment of a cell population stably expressing GaLV10A1ΔR. SLC20A1 deletion led to a decrease in cell growth, however it was restored by SLC20A2 complementation. Altogether, this thesis supports the viral receptor knock-out strategy to develop new cell lines to express glycoproteins with fusogenicity-derived cytotoxicity and provides new insights that will help improve this strategy. This work contributes to the state-of-the-art of the lentiviral vector-based gene therapy, providing new insights that will improve the development of constitutive producer cells in the near future.